Osteoclasts are large, multinucleated cells that play a central role in bone resorption. They are derived from hematopoietic precursors in response to a number of regulatory factors, the most important of which is RANKL. RANKL is both necessary and sufficient for osteoclastogenesis, although factors such as M-CSF, TGFb, inflammatory cytokines and prostaglandins are also important in the events associated with this process. RANKL is synthesized in a number of cell types, including those of the osteoblast lineage. Studies both in vitro and in vivo indicate that RANKL mRNA expression is regulated physiologically by key bone remodeling hormones such as PTH and 1,25(OH)2D3 as well as by cytokines such as IL-1, TNFa, IL-6 and the prostaglandin PGE2. Aberrant production and/or expression of any one these modulators has been implicated in RANKL overexpression, enhanced bone resorption and osteolytic or osteoporotic disease. Despite considerable physiologic and pathologic insight, little is known of the molecular mechanisms that control the transcriptional output of RANKL from support cells. Initial studies, however, suggest that the mechanisms responsible are likely to be highly complex. In view of the critical impact of RANKL on bone resorption, we propose to define key mechanisms instrumental to RANKL gene expression at the molecular level. Aim 1: To define the molecular events associated with activation of the RANKL gene by VDR, CREB, and STATS at the RANKL distal control region (RL- DCR) located 76 kb upstream of the mouse RankL transcriptional start site (TSS). The relevance of this site will be confirmed using a genetically altered mouse model. Aim 2: To evaluate the contribution of four additional regulatory domains located upstream of the mouse RankL gene TSS at -16, -22, -60 and -69 kb. The contribution of these sites to the regulation of RankL gene expression will be examined through preparation of a unique mouse model. Aim 3: To assess the consequence of transcription factor occupancy within the RankL gene upstream region on chromatin organization, modification and structure, and to determine the function of RNA polymerase II which appears specifically at these sites in response to activation. These studies will provide important detail into the mechanism by which calciotropic hormones and cytokines regulate RANKL expression, a gene whose product is central to bone resorption.